Zinc is an essential nutrient needed for many cellular processes, however, excess zinc leads to cell death. One mechanism to regulate cellular zinc homeostasis involves the Zrt-, Irt-like, Protein (ZIP) family of zinc transporters which function to import zinc into the cytoplasm. This research proposal addresses the role of the human transporter Zip13 (hZip13) in maintaining zinc homeostasis in human cells. To address the role of hZip13 in human cells, immunofluorescence microscopy will be used to localize hZip13 to its subcellular site of action in basal conditions in both fibroblasts and polarized epithelial cells. Furthermore, hZip13 localization will be monitored in high and low zinc conditions to determine if zinc regulates hZip13 localization. With basic localization information about hZip13, the function of hZip13 will be analyzed using fluorescent chemosensors to monitor zinc mobilization into the cytoplasm. Reporter assays will also be used to monitor changes in cytosolic, endoplasmic reticulum, and secretory pathway zinc status. To characterize the mechanism upregulating hZip13 mRNA expression levels in low zinc, sequence elements in the promoter region of SLC39A13, the gene which encodes for the hZipl 3 protein, or the mRNA transcript will be analyzed. Nuclear run-on assays will be used to determine if the rate of transcript initiation changes in low cellular zinc or whether the half-life of hZip13 mRNA half-life changes in response to low cellular zinc. Should transcription initiation control hZip13 mRNA abundance, the transcription start site will be determined using rapid amplification of cDNA ends and confirmed using S1 nuclease protection assays. Once the transcription initiation site is known, the hZip13 promoter region will be dissected using a luciferase reporter assay where systematic deletions of the hZipl 3 promoter fused to the luciferase gene will be expressed in human cells in low zinc. In contrast, should mRNA stability control transcript levels, analogous experiments will be done to identify the sequence element important for regulating transcript level in low zinc. The identification of the sequence elements controlling hZip13 mRNA expression in low zinc will eventually lead to determining the molecular mechanisms involved in regulating zinc homeostasis in low zinc conditions. [unreadable] [unreadable] The proposed study will determine the function of the human zinc transporter Zip13 in human cells and the role it plays in maintaining zinc homeostasis. This proposal will begin to identify the genetic mechanisms controlling human Zip13 in response to low cellular zinc providing clues to how human cells regulate zinc metabolism. [unreadable] [unreadable] [unreadable]